Nucleotide sequences coding for the pknB gene

ABSTRACT

An isolated polynucleotide which contains a polynucleotide sequence selected from the group comprising:
         (a) a polynucleotide which is at least 70% identical to a polynucleotide coding for a polypeptide containing the amino acid sequence of SEQ ID No. 2,   (b) a polynucleotide coding for a polypeptide containing an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID No. 2,   (c) a polynucleotide which is complementary to the polynucleotides of (a) or (b), and   (d) a polynucleotide containing at least 15 consecutive nucleotides of the polynucleotide sequence of (a), (b) or (c),   and a fermentation process for the preparation of L-amino acids using corynebacteria in which at least the pknB gene is amplified, and to the use, as hybridization probes, of polynucleotides containing the sequences according to the invention.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims benefit to U.S. Provisional Application Ser. No.60/297,250, filed on Jun. 12, 2001, and incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates nucleotide sequences from corynebacteriacoding for the pknB gene and a fermentation process for the preparationof amino acids using bacteria in which the endogenous pknB gene isamplified.

2. State of the Art

L-Amino acids, especially L-lysine, are used in human medicine, in thepharmaceutical industry, in the food industry, and, in particular, inanimal nutrition. It is known that amino acids are prepared by thefermentation of strains of corynebacteria, especially Corynebacteriumglutamicum. Because of their great importance, attempts are constantlybeing made to improve these preparative processes. Improvements to theprocesses may relate to measures involving the fermentation technology,e.g. stirring and oxygen supply, or the composition of the nutrientmedia, e.g. the sugar concentration during fermentation, or the work-upto the product form, e.g. by ion exchange chromatography, or theintrinsic productivity characteristics of the microorganism itself.

The productivity characteristics of these microorganisms are improved byusing methods of mutagenesis, selection and mutant choice to givestrains which are resistant to antimetabolites or auxotrophic formetabolites important in regulation, and produce amino acids.

Methods of recombinant DNA technology have also been used for some yearsto improve L-amino acid-producing strains of Corynebacterium byamplifying individual amino acid biosynthesis genes and studying theeffect on amino acid production. However, there remains a need forimproved methods of producing L-amino acids.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide novel measures forimproving the preparation of amino acids by fermentation.

It is another object of the present invention to provide nucleic acidsequences which are useful for the production of amino acids.

Accordingly, the present invention provides an isolated polynucleotidefrom corynebacteria which contains a polynucleotide sequence coding forthe pknB gene and is selected from the following group:

(a) a polynucleotide which is at least 70% identical to a polynucleotidecoding for a polypeptide containing the amino acid sequence of SEQ IDNo. 2,

(b) a polynucleotide coding for a polypeptide containing an amino acidsequence which is at least 70% identical to the amino acid sequence ofSEQ ID No. 2,

(c) a polynucleotide which is complementary to the polynucleotides of(a) or (b), and

(d) a polynucleotide containing at least 15 consecutive nucleotides ofthe polynucleotide sequence of (a), (b) or (c),

where the polypeptide preferably has the activity of protein kinase B.

The present invention also provides the above-mentioned polynucleotide,which is preferably a replicatable DNA containing:

(i) the nucleotide sequence shown in SEQ ID No. 1, or

(ii) at least one sequence corresponding to sequence (i) within thedegeneracy of the genetic code, or

(iii) at least one sequence which hybridizes with the sequencecomplementary to sequence (i) or (ii), and optionally

(iv) neutral sense mutations in (i).

The present invention also provides:

-   -   a replicatable polynucleotide, especially DNA, containing the        nucleotide sequence as shown in SEQ ID No. 1,    -   a polynucleotide coding for a polypeptide containing the amino        acid sequence as shown in SEQ ID No. 2,    -   a vector containing the polynucleotide according to the        invention, especially a shuttle vector or plasmid vector, and    -   corynebacteria which contain the vector or in which the endogeny        pknB gene is amplified.

The present invention also provides a fermentation process for thepreparation of an L-amino acid, comprising:

(a) fermenting corynebacteria which produce the L-amino acid in amedium, wherein at least the pknB gene or nucleotide sequences codingtherefor are amplified in the corynebacteria,

(b) enriching the L-amino acid in the medium or in the cells of thecorynebacteria, and

(c) isolating the L-amino acid.

The present invention additionally provides polynucleotides consistingsubstantially of a polynucleotide sequence which are obtainable byscreening, by means of hybridization, of an appropriate gene library ofa corynebacterium, containing the complete gene or parts thereof, with aprobe containing the sequence of the polynucleotide of the inventionaccording to SEQ ID No. 1 or a fragment thereof, and by isolation of thepolynucleotide sequence.

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

When L-amino acids or amino acids are mentioned hereafter, this isunderstood to refer to one or more amino acids, including their salts,selected from the group comprising L-asparagine, L-threonine, L-serine,L-glutamate, L-glycine, L-alanine, L-cysteine, L-valine, L-methionine,L-isoleucine, L-leucine, L-tyrosine, L-phenylalanine, L-histidine,L-lysine, L-tryptophan and L-arginine. L-Lysine is particularlypreferred.

When L-lysine or lysine is mentioned hereafter, this is understood torefer to not only the bases but also the salts, e.g. lysinemonohydrochloride or lysine sulfate.

As hybridization probes for RNA, cDNA and DNA, polynucleotidescontaining the sequences according to the invention are suitable forisolating the full length of nucleic acids, or polynucleotides or genes,coding for protein kinase B, or for isolating nucleic acids, orpolynucleotides or genes, whose sequence exhibits a high degree ofsimilarity to the sequence of the pknB gene. They are also suitable forincorporation into so-called arrays, micro-arrays or DNA chips fordetecting and determining the corresponding polynucleotides.

Polynucleotides containing the sequences according to the invention arefurther suitable as primers for the preparation, by the polymerase chainreaction (PCR), of DNA of genes coding for protein kinase B.

Such oligonucleotides serving as probes or primers contain at least 25,26, 27, 28, 29 or 30, preferably at least 20, 21, 22, 23 or 24 and veryparticularly preferably at least 15, 16, 17, 18 or 19 consecutivenucleotides. Oligonucleotides with a length of at least 31, 32, 33, 34,35, 36, 37, 38, 39 or 40 or at least 41, 42, 43, 44, 45, 46, 47, 48, 49or 50 nucleotides are also suitable. Oligonucleotides with a length ofat least 100, 150, 200, 250 or 300 nucleotides are also suitable.

As used herein, the term “isolated” refers to a material, e.g., anucleic acid sequence, separated from its natural environment.

“Polynucleotide” refers in general to polyribonucleotides andpolydeoxyribonucleotides, it being possible for the RNAs or DNAs inquestion to be unmodified or modified.

The polynucleotides according to the invention include a polynucleotideaccording to SEQ ID No. 1 or a fragment prepared therefrom, as well aspolynucleotides which are in particular at least 70% to 80%, preferablyat least 81% to 85%, particularly preferably at least 86% to 90% andvery particularly preferably at least 91%, 93%, 95%, 97% or 99%identical to the polynucleotide according to SEQ ID No. 1 or a fragmentprepared therefrom.

“Polypeptides” are understood as meaning peptides or proteins containingtwo or more amino acids bonded via peptide links.

The polypeptides according to the invention include a polypeptideaccording to SEQ ID No. 2, especially those with the biological activityof protein kinase B and also those which are at least 70% to 80%,preferably at least 81% to 85%, particularly preferably at least 86% to90% and very particularly preferably at least 91%, 93%, 95%, 97% or 99%identical to the polypeptide according to SEQ ID No. 2, and having theactivity of protein kinase B.

The invention further relates to a fermentation process for thepreparation of amino acids selected from the group comprisingL-asparagine, L-threonine, L-serine, L-glutamate, L-glycine, L-alanine,L-cysteine, L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine,L-phenylalanine, L-histidine, L-lysine, L-tryptophan and L-arginine,using corynebacteria which, in particular, already produce amino acidsand in which the nucleotide sequences coding for the pknB gene areamplified and, in particular, overexpressed.

In this context the term “enhancement” describes the increase in theintracellular activity, in a microorganism, of one or more enzymes whichare coded for by the appropriate DNA, for example by increasing the copynumber of the gene(s) or allele(s), using a strong promoter or using agene or allele coding for an appropriate enzyme with a high activity,and optionally combining these measures.

By enhancement measures, in particular over-expression, the activity orconcentration of the corresponding protein is in general increased by atleast 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to amaximum of 1000% or 2000%, based on the starting microorganism.

The microorganisms provided by the present invention can produce L-aminoacids from glucose, sucrose, lactose, fructose, maltose, molasses,starch or cellulose or from glycerol and ethanol. The microorganisms canbe representatives of corynebacteria, especially of the genusCorynebacterium. The species Corynebacterium glutamicum may be mentionedin particular in the genus Corynebacterium, which is known to thoseskilled in the art for its ability to produce L-amino acids.

The following known wild-type strains:

-   -   Corynebacterium glutamicum ATCC13032    -   Corynebacterium acetoglutamicum ATCC15806    -   Corynebacterium acetoacidophilum ATCC13870    -   Corynebacterium thermoaminogenes FERM BP-1539    -   Corynebacterium melassecola ATCC17965    -   Brevibacterium flavum ATCC14067    -   Brevibacterium lactofermentum ATCC13869 and    -   Brevibacterium divaricatum ATCC14020        and L-amino acid-producing mutants or strains prepared        therefrom, are particularly suitable strains of the genus        Corynebacterium, especially of the species Corynebacterium        glutamicum (C. glutamicum).

The novel pknB gene of C. glutamicum coding for the enzyme proteinkinase B (EC 2.7.1.37) has been isolated using the procedures describedherein.

The first step in isolating the pknB gene or other genes of C.glutamicum is to construct a gene library of this microorganism inEscherichia coli (E. coli). The construction of gene libraries isdocumented in generally well-known textbooks and manuals. Examples whichmay be mentioned are the textbook by Winnacker entitled From Genes toClones, Introduction to Gene Technology (Verlag Chemie, Weinheim,Germany, 1990) or the manual by Sambrook et al. entitled MolecularCloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press,1989). A very well-known gene library is that of the E. coli K-12 strainW3110, which was constructed by Kohara et al. (Cell 50, 495–508 (1987))in λ vectors. Bathe et al. (Molecular and General Genetics 252, 255–265,1996) describe a gene library of C. glutamicum ATCC13032, which wasconstructed using cosmid vector SuperCos I (Wahl et al., 1987,Proceedings of the National Academy of Sciences USA 84, 2160–2164) inthe E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic AcidsResearch 16, 1563–1575). Börmann et al. (Molecular Microbiology 6(3),317–326 (1992)) in turn describe a gene library of C. glutamicumATCC13032 using cosmid pHC79 (Hohn and Collins, Gene 11, 291–298(1980)).

A gene library of C. glutamicum in E. coli can also be constructed usingplasmids like pBR322 (Bolivar, Life Sciences 25, 807–818 (1979)) or pUC9(Vieira et al., 1982, Gene 19, 259–268). Restriction- andrecombination-defective E. coli strains are particularly suitable ashosts, an example being the strain DH5αmcr, which has been described byGrant et al. (Proceedings of the National Academy of Sciences USA 87(1990) 4645–4649). The long DNA fragments cloned with the aid of cosmidscan then in turn be subcloned into common vectors suitable forsequencing, and subsequently sequenced, e.g. as described by Sanger etal. (Proceedings of the National Academy of Sciences of the UnitedStates of America 74, 5463–5467, 1977).

The DNA sequences obtained can then be examined with known algorithms orsequence analysis programs, e.g. that of Staden (Nucleic Acids Research14, 217–232 (1986)), that of Marck (Nucleic Acids Research 16, 1829–1836(1988)) or the GCG program of Butler (Methods of Biochemical Analysis39, 74–97 (1998)).

The novel DNA sequence of C. glutamicum coding for the pknB gene wasfound and, as SEQ ID No. 1, forms part of the present invention.Furthermore, the amino acid sequence of the corresponding protein wasderived from the DNA sequence by the methods described above. Theresulting amino acid sequence of the pknB gene product is shown in SEQID No. 2.

Coding DNA sequences which result from SEQ ID No. 1 due to thedegeneracy of the genetic code also within the scope of the presentinvention. Likewise, DNA sequences which hybridize with SEQ ID No. 1 orportions of SEQ ID No. 1 form part of the invention. Furthermore,conservative amino acid exchanges, e.g. the exchange of glycine foralanine or of aspartic acid for glutamic acid in proteins, are known tothose skilled in the art as “sense mutations”, which do not cause afundamental change in the activity of the protein, i.e. they areneutral. It is also known that changes at the N- and/or C-terminus of aprotein do not substantially impair its function or may even stabilizeit. Those skilled in the art will find information on this subject inBen-Bassat et al. (Journal of Bacteriology 169, 751–757 (1987)), O'Reganet al. (Gene 77, 237–251 (1989)), Sahin-Toth et al. (Protein Sciences 3,240–247 (1994)) and Hochuli et al. (Bio/Technology 6, 1321–1325 (1988)),inter alia, and in well-known textbooks on genetics and molecularbiology. Amino acid sequences which correspondingly result from SEQ IDNo. 2 also form part of the invention.

Likewise, DNA sequences which hybridize with SEQ ID No. 1 or portions ofSEQ ID No. 1 are within the scope of the present invention. Finally, DNAsequences which are prepared by the polymerase chain reaction (PCR)using primers resulting from SEQ ID No. 1 form part of the invention.Such oligonucleotides typically have a length of at least 15nucleotides.

Those skilled in the art can find instructions on the identification ofDNA sequences by means of hybridization in the manual entitled “The DIGSystem User's Guide for Filter Hybridization” from Boehringer MannheimGmbH (Mannheim, Germany, 1993) and in Liebl et al. (InternationalJournal of Systematic Bacteriology (1991) 41, 255–260), inter alia.Hybridization takes place under stringent conditions; in other words,only hybrids for which the probe and the target sequence, i.e. thepolynucleotides treated with the probe, are at least 70% identical areformed. It is known that the stringency of hybridization, including thewashing steps, is influenced or determined by varying the buffercomposition, the temperature and the salt concentration. Thehybridization reaction is preferably carried out under relatively lowstringency compared with the washing steps (Hybaid Hybridisation Guide,Hybaid Limited, Teddington, UK, 1996).

The hybridization reaction can be carried out for example using a 5×SSCbuffer at a temperature of approx. 50° C.–68° C., it also being possiblefor probes to hybridize with polynucleotides which are less than 70%identical to the sequence of the probe. Such hybrids are less stable andare removed by washing under stringent conditions. This can be achievedfor example by lowering the salt concentration to 2×SSC and subsequentlyto 0.5×SSC if necessary (The DIG System User's Guide for FilterHybridization, Boehringer Mannheim, Mannheim, Germany, 1995), thetemperature being adjusted to approx. 50° C.–68° C. It is possible tolower the salt concentration to 0.1×SSC if necessary. By raising thehybridization temperature in approx. 1–2° C. steps from 50° C. to 68°C., it is possible to isolate polynucleotide fragments which are e.g. atleast 70%, at least 80% or at least 90% to 95% identical to the sequenceof the probe used. Further instructions on hybridization arecommercially available in the form of kits (e.g. DIG Easy Hyb from RocheDiagnostics GmbH, Mannheim, Germany, Catalog No. 1603558).

Those skilled in the art can find instructions on the amplification ofDNA sequences with the aid of the polymerase chain reaction (PCR) in themanual by Gait entitled Oligonucleotide Synthesis: A Practical Approach(IRL Press, Oxford, UK, 1984) and in Newton and Graham: PCR (SpektrumAkademischer Verlag, Heidelberg, Germany, 1994), inter alia.

It has been found that, after overexpression of the pknB gene, theproduction of amino acids by corynebacteria is improved.

Overexpression can be achieved by increasing the copy number of theappropriate genes or mutating the promoter and regulatory region or theribosome binding site located upstream from the structural gene.Expression cassettes incorporated upstream from the structural gene workin the same way. Inducible promoters additionally make it possible toincrease the expression in the course of the production of amino acid byfermentation. Measures for prolonging the life of the mRNA also improvethe expression. Furthermore, the enzyme activity is also enhanced bypreventing the degradation of the enzyme protein. The genes or geneconstructs can either be located in plasmids of variable copy number orintegrated and amplified in the chromosome. Alternatively, it is alsopossible to achieve overexpression of the genes in question by changingthe composition of the media and the culture technique.

Those skilled in the art can find relevant instructions in Martin et al.(Bio/Technology 5, 137–146 (1987)), Guerrero et al. (Gene 138, 35–41(1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428–430 (1988)),Eikmanns et al. (Gene 102, 93–98 (1991)), EP 0 472 869, U.S. Pat. No.4,601,893, Schwarzer and Pühler (Bio/Technology 9, 84–87 (1991)),Reinscheid et al. (Applied and Environmental Microbiology 60, 126–132(1994)), LaBarre et al. (Journal of Bacteriology 175, 1001–1007 (1993)),WO 96/15246, Malumbres et al. (Gene 134, 15–24 (1993)), JP-A-10-229891,Jensen and Hammer (Biotechnology and Bioengineering 58, 191–195 (1998))and Makrides (Microbiological Reviews 60, 512–538 (1996)), inter alia,and in well-known textbooks on genetics and molecular biology.

For amplification, the pknB gene according to the invention has beenoverexpressed for example with the aid of episomal plasmids. Suitableplasmids are those which are replicated in corynebacteria. Numerousknown plasmid vectors, e.g. pZ1 (Menkel et al., Applied andEnvironmental Microbiology (1989) 64, 549–554), pEKEx1 (Eikmanns et al.,Gene 102, 93–98 (1991)) or pHS2-1 (Sonnen et al., Gene 107, 69–74(1991)), are based on cryptic plasmids pHM1519, pBL1 or pGA1. Otherplasmid vectors, e.g. those based on pCG4 (U.S. Pat. No. 4,489,160),pNG2 (Serwold-Davis et al., FEMS Microbiology Letters 66, 119–124(1990)) or pAG1 (U.S. Pat. No. 5,158,891), can be used in the same way.

Other suitable plasmid vectors are those which make it possible to usethe gene amplification process by integration into the chromosome, asdescribed for example by Reinscheid et al. (Applied and EnvironmentalMicrobiology 60, 126–132 (1994)) for the duplication or amplification ofthe hom-thrB operon. In this method the complete gene is cloned into aplasmid vector which can replicate in a host (typically E. coli), butnot in C. glutamicum. Examples of suitable vectors are pSUP301 (Simon etal., Bio/Technology 1, 784–791 (1983)), pK18mob or pK19mob (Schäfer etal., Gene 145, 69–73 (1994)), pGEM-T (Promega Corporation, Madison,Wis., USA), pCR2.1-TOPO (Shuman (1994), Journal of Biological Chemistry269, 32678–84; U.S. Pat. No. 5,487,993), pCR®Blunt (Invitrogen,Groningen, The Netherlands; Bernard et al., Journal of Molecular Biology234, 534–541 (1993)), pEM1 (Schrumpf et al., 1991, Journal ofBacteriology 173, 4510–4516) or pBGS8 (Spratt et al., 1986, Gene 41,337–342). The plasmid vector containing the gene to be amplified is thentransferred to the desired strain of C. glutamicum by conjugation ortransformation. The method of conjugation is described for example inSchäfer et al. (Applied and Environmental Microbiology 60, 756–759(1994)). Methods of transformation are described for example inThierbach et al. (Applied Microbiology and Biotechnology 29, 356–362(1988)), Dunican and Shivnan (Bio/Technology 7, 1067–1070 (1989)) andTauch et al. (FEMS Microbiological Letters 123, 343–347 (1994)). Afterhomologous recombination by means of a crossover event, the resultingstrain contains at least two copies of the gene in question.

It has also been found that amino acid exchanges in the section betweenposition 581 and position 587 of the amino acid sequence of proteinkinase B, shown in SEQ ID No. 2, improve the production of amino acids,especially lysine, by corynebacteria. Preferably, L-proline in position584 is exchanged for any other proteogenic amino acid except L-proline,preferably for L-serine or L-threonine and very particularly preferablyfor L-serine.

SEQ ID No. 3 shows the base sequence of the pknB-1547 allele containedin the strain DM1547. The pknB-1547 allele codes for a protein whoseamino acid sequence is shown in SEQ ID No. 4. The protein containsL-serine in position 584. The DNA sequence of the pknB-1547 allele (SEQID No. 3) contains the base thymine in place of the base cytosinecontained in the pknB wild-type gene (SEQ ID No. 1) in position 2343.

Mutagenesis can be carried out by conventional methods using mutagenicsubstances such as N-methyl-N′-nitro-N-nitrosoguanidine or ultravioletlight. Mutagenesis can also be carried out using in vitro methods suchas treatment with hydroxylamine (Miller, J. H.: A Short Course inBacterial Genetics. A Laboratory Manual and Handbook for Escherichiacoli and Related Bacteria, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, 1992) or mutagenic oligonucleotides (T. A. Brown:Gentechnologie für Einsteiger (Gene Technology for Beginners), SpektrumAkademischer Verlag, Heidelberg, 1993), or the polymerase chain reaction(PCR) as described in the manual by Newton and Graham (PCR, SpektrumAkademischer Verlag, Heidelberg, 1994).

The corresponding alleles or mutations are sequenced and introduced intothe chromosome by the method of gene replacement, for example asdescribed in Peters-Wendisch et al. (Microbiology 144, 915–927 (1998))for the pyc gene of C. glutamicum, in Schäfer et al. (Gene 145, 69–73(1994)) for the hom-thrB gene region of C. glutamicum or in Schäfer etal. (Journal of Bacteriology 176, 7309–7319 (1994)) for the cg1 generegion of C. glutamicum. The corresponding alleles or the associatedproteins can optionally be amplified in turn.

In addition it can be advantageous for the production of L-amino acidsto amplify and, in particular, overexpress not only the pknB gene butalso one or more enzymes of the particular biosynthetic pathway, theglycolysis, the anaplerosis, the citric acid cycle, the pentosephosphate cycle or the amino acid export, and optionally regulatoryproteins.

Thus, for the production of L-amino acids, one or more genes selectedfrom the following group can be amplified and, in particular,overexpressed in addition to amplification of the endogene pknB gene:

the dapA gene coding for dihydrodipicolinate synthase (EP-B-0 197 335),

-   -   the gap gene coding for glyceraldehyde 3-phosphate dehydrogenase        (Eikmanns (1992), Journal of Bacteriology 174, 6076–6086),    -   the tpi gene coding for triose phosphate isomerase (Eikmanns        (1992), Journal of Bacteriology 174, 6076–6086),    -   the pgk gene coding for 3-phosphoglycerate kinase (Eikmanns        (1992), Journal of Bacteriology 174, 6076–6086),    -   the zwf gene coding for glucose-6-phosphate dehydrogenase        (JP-A-09224661),    -   the pyc gene coding for pyruvate carboxylase (DE-A-198 31 609),    -   the lysC gene coding for a feedback-resistant aspartate kinase        (Accession no. P26512; EP-B-0387527; EP-A-0699759),    -   the lysE gene coding for lysine export (DE-A-195 48 222),    -   the hom gene coding for homoserine dehydrogenase (EP-A-0131171),    -   the ilvA gene coding for threonine dehydratase (Möckel et al.,        Journal of Bacteriology (1992) 8065–8072) or the ilvA(Fbr)        allele coding for a feedback-resistant threonine dehydratase        (Möckel et al. (1994), Molecular Microbiology 13, 833–842),    -   the ilvBN gene coding for acetohydroxy acid synthase        (EP-B-0356739),    -   the ilvD gene coding for dihydroxy acid dehydratase (Sahm and        Eggeling (1999), Applied and Environmental Microbiology 65,        1973–1979), or    -   the zwa1 gene coding for the Zwa1 protein (DE 199 59 328.0, DSM        13115).

In addition to amplification of the pknB gene, it can also beadvantageous for the production of L-amino acids to attenuate one ormore genes selected from the following group:

-   -   the pck gene coding for phosphoenol pyruvate carboxykinase (DE        199 50 409.1, DSM13047),    -   the pgi gene coding for glucose-6-phosphate isomerase (U.S. Ser.        No. 09/396,478, DSM12969),    -   the poxB gene coding for pyruvate oxidase (DE 199 51 975.7,        DSM13114), or    -   the zwa2 gene coding for the Zwa2 protein (DE 199 59 327.2,        DSM13113),        and, in particular, to reduce the expression.

In this context the term “attenuation” describes the reduction orswitching-off of the intracellular activity, in a microorganism, of oneor more enzymes (proteins) which are coded for by the appropriate DNA,for example by using a weak promoter or using a gene or allele codingfor an appropriate enzyme with a low activity, or inactivating theappropriate gene or enzyme (protein), and optionally combining thesemeasures.

By attenuation measures, the activity or concentration of thecorresponding protein is in general reduced to 0 to 50%, 0 to 25%, 0 to10% or 0 to 5% of the activity or concentration of the wild-typeprotein.

It can also be advantageous for the production of amino acids not onlyto overexpress the pknB gene but also to switch off unwanted secondaryreactions (Nakayama: “Breeding of Amino Acid Producing Micro-organisms”,in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek(eds.), Academic Press, London, UK, 1982).

The microorganisms prepared according to the invention are also providedby the invention and can be cultivated for the production of amino acidscontinuously or discontinuously by the batch process, the fed batchprocess or the repeated fed batch process. A summary of knowncultivation methods is described in the textbook by Chmiel(Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik (BioprocessTechnology 1. Introduction to Bioengineering) (Gustav Fischer Verlag,Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren undperiphere Einrichtungen (Bioreactors and Peripheral Equipment) (ViewegVerlag, Brunswick/Wiesbaden, 1994)).

The culture medium to be used must appropriately meet the demands of theparticular strains. Descriptions of culture media for variousmicroorganisms can be found in “Manual of Methods for GeneralBacteriology” of the American Society for Bacteriology (Washington D.C.,USA, 1981).

Carbon sources which can be used are sugars and carbohydrates, e.g.glucose, sucrose, lactose, fructose, maltose, molasses, starch andcellulose, oils and fats, e.g. soybean oil, sunflower oil, groundnut oiland coconut fat, fatty acids, e.g. palmitic acid, stearic acid andlinoleic acid, alcohols, e.g. glycerol and ethanol, and organic acids,e.g. acetic acid. These substances can be used individually or as amixture.

Nitrogen sources which can be used are organic nitrogen-containingcompounds such as peptones, yeast extract, meat extract, malt extract,corn steep liquor, soybean flour and urea, or inorganic compounds suchas ammonium sulfate, ammonium chloride, ammonium phosphate, ammoniumcarbonate and ammonium nitrate. The nitrogen sources can be usedindividually or as a mixture.

Phosphorus sources which can be used are phosphoric acid, potassiumdihydrogenphosphate or dipotassium hydrogenphosphate or thecorresponding sodium salts. The culture medium must also contain metalsalts, e.g. magnesium sulfate or iron sulfate, which are necessary forgrowth. Finally, essential growth-promoting substances such as aminoacids and vitamins can be used in addition to the substances mentionedabove. Suitable precursors can also be added to the culture medium. Thefeed materials can be added to the culture all at once or fed inappropriately during cultivation.

The pH of the culture is controlled by the appropriate use of basiccompounds such as sodium hydroxide, potassium hydroxide, ammonia oraqueous ammonia, or acidic compounds such as phosphoric acid or sulfuricacid. Foaming can be controlled using antifoams such as fatty acidpolyglycol esters. The stability of plasmids can be maintained by addingsuitable selectively acting substances, e.g. antibiotics, to the medium.Aerobic conditions are maintained by introducing oxygen oroxygen-containing gaseous mixtures, e.g. air, into the culture. Thetemperature of the culture is normally 20° C. to 45° C. and preferably25° C. to 40° C. The culture is continued until the formation of thedesired product has reached a maximum. This objective is normallyachieved within 10 hours to 160 hours.

Methods of determining L-amino acids are well-known to those skilled inthe art. They can be analyzed for example by ion exchange chromatographywith subsequent ninhydrin derivation, as described by Spackman et al.(Analytical Chemistry 30 (1958) 1190), or by reversed phase HPLC, asdescribed by Lindroth et al. (Analytical Chemistry (1979) 51,1167–1174).

A pure culture of the Corynebacterium glutamicum strain DM1547 wasdeposited as DSM13994 in the Deutsche Sammlung für Mikroorganismen undZellkulturen (German Collection of Microorganisms and Cell Cultures(DSMZ), Brunswick, Germany) on 16 Jan. 2001 under the terms of theBudapest Treaty.

The fermentation process according to the invention is used for thepreparation of amino acids.

Another aspect of the present invention is a method of identifyingnucleic acids which code for protein kinase B or have a high degree ofsimilarity to the sequence of the pknB gene, comprising:

-   -   contacting a sample with the polynucleotide of claim 1 under        conditions suitable for the polynucleotide to hydridize to        another nucleic acid which codes for protein kinase B or have a        high degree of similarity to the sequence of the pknB gene. In        one embodiment, the another nucleic acid referred to above is        RNA, cDNA, or DNA. In another embodiment, the method is        conducted on an array, micro-array, or DNA chip.

EXAMPLES

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

The isolation of plasmid DNA from Escherichia coli and all thetechniques of restriction, Klenow treatment and alkaline phosphatasetreatment were carried out according to Sambrook et al. (MolecularCloning. A Laboratory Manual (1989), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., USA). Methods of transformingEscherichia coli are also described in this manual.

The composition of common nutrient media, such as LB or TY medium, canalso be found in the manual by Sambrook et al.

Example 1 Preparation of a Genomic Cosmid Gene Library fromCorynebacterium glutamicum ATCC13032

Chromosomal DNA from Corynebacterium glutamicum ATCC13032 was isolatedas described in Tauch et al. (1995, Plasmid 33, 168–179) and partiallycleaved with the restriction enzyme Sau3AI (Amersham Pharmacia,Freiburg, Germany, product description Sau3AI, code no. 27-0913-02). TheDNA fragments were dephosphorylated with shrimp alkaline phosphatase(Roche Diagnostics GmbH, Mannheim, Germany, product description SAP,code no. 1758250). The DNA of cosmid vector SuperCos1 (Wahl et al.(1987), Proceedings of the National Academy of Sciences USA 84,2160–2164), obtained from Stratagene (La Jolla, USA, product descriptionSuperCos1 Cosmid Vector Kit, code no. 251301), was cleaved with therestriction enzyme XbaI (Amersham Pharmacia, Freiburg, Germany, productdescription XbaI, code no. 27-0948-02) and also dephosphorylated withshrimp alkaline phosphatase.

The cosmid DNA was then cleaved with the restriction enzyme BamHI(Amersham Pharmacia, Freiburg, Germany, product description BamHI, codeno. 27-0868-04). The cosmid DNA treated in this way was mixed with thetreated ATCC13032 DNA and the mixture was treated with T4 DNA ligase(Amersham Pharmacia, Freiburg, Germany, product description T4 DNAligase, code no. 27-0870-04). The ligation mixture was then packagedinto phages using Gigapack II XL Packing Extract (Stratagene, La Jolla,USA, product description Gigapack II XL Packing Extract, code no.200217).

For infection of the E. coli strain NM554 (Raleigh et al., 1988, NucleicAcid Research 16, 1563-1575), the cells were taken up in 10 mM MgSO₄ andmixed with an aliquot of the phage suspension. Infection and titering ofthe cosmid library were carried out as described in Sambrook et al.(1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor), thecells being plated on LB agar (Lennox, 1955, Virology 1, 190) containing100 mg/l of ampicillin. After incubation overnight at 37° C.,recombinant single clones were selected.

Example 2 Isolation and Sequencing of the pknB Gene

The cosmid DNA of a single colony was isolated with the Qiaprep SpinMiniprep Kit (product no. 27106, Qiagen, Hilden, Germany) in accordancewith the manufacturer's instructions and partially cleaved with therestriction enzyme Sau3AI (Amersham Pharmacia, Freiburg, Germany,product description Sau3AI, product no. 27-0913-02). The DNA fragmentswere dephosphorylated with shrimp alkaline phosphatase (RocheDiagnostics GmbH, Mannheim, Germany, product description SAP, productno. 1758250). After separation by gel electrophoresis, the cosmidfragments in the size range from 1500 to 2000 bp were isolated with theQiaExII Gel Extraction Kit (product no. 20021, Qiagen, Hilden, Germany).

The DNA of sequencing vector pZero-1, obtained from Invitrogen(Groningen, The Netherlands, product description Zero Background CloningKit, product no. K2500-01), was cleaved with the restriction enzymeBamHI (Amersham Pharmacia, Freiburg, Germany, product description BamHI,product no. 27-0868-04). Ligation of the cosmid fragments intosequencing vector pZero-1 was carried out as described in Sambrook etal. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor),the DNA mixture being incubated overnight with T4 ligase (PharmaciaBiotech, Freiburg, Germany). This ligation mixture was then introducedinto the E. coli strain DH5αMCR (Grant, 1990, Proceedings of theNational Academy of Sciences USA 87, 4645–4649) by electroporation(Tauch et al. 1994, FEMS Microbiol. Letters 123, 343–7) and plated on LBagar (Lennox, 1955, Virology 1, 190) containing 50 mg/l of zeocin.

Plasmid preparation of the recombinant clones was carried out withBiorobot 9600 (product no. 900200, Qiagen, Hilden, Germany). Sequencingwas carried out by the dideoxy chain termination method of Sanger et al.(1977, Proceedings of the National Academy of Sciences USA 74,5463–5467) with modifications by Zimmermann et al. (1990, Nucleic AcidsResearch 18, 1067). The “RR dRhodamin Terminator Cycle Sequencing Kit”from PE Applied Biosystems (product no. 403044, Weiterstadt, Germany)was used. Separation by gel electrophoresis and analysis of thesequencing reaction were carried out in a “Rotiphorese NFacrylamide/bisacrylamide” gel (29:1) (product no. A124.1, Roth,Karlsruhe, Germany) with the “ABI Prism 377” sequencer from PE AppliedBiosystems (Weiterstadt, Germany).

The raw sequence data obtained were then processed using the Stadenprogramming package (1986, Nucleic Acids Research 14, 217–231), version97-0. The individual sequences of the pZero-1 derivatives were assembledinto a cohesive contig. Computer-assisted coding region analysis wasperformed with the XNIP program (Staden, 1986, Nucleic Acids Research14, 217–231).

The nucleotide sequence obtained is shown in SEQ ID No. 1. Analysis ofthe nucleotide sequence gave an open reading frame of 1884 base pairs,which was called the pknB gene. The pknB gene codes for a protein of 627amino acids.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

All of the publications cited above are incorporated herein byreference.

This application is based, inter alia, on German Patent ApplicationSerial No. 100 44 912.3, filed on Sep. 12, 2000; and German PatentApplication Serial No. 101 20 095.1, filed on Apr. 25, 2001, both ofwhich are incorporated herein by reference.

1. An isolated polynucleotide consisting of at least 26 consecutivenucleotides of SEQ ID NO:1 or the full complement of SEQ ID NO:1,wherein the isolated polynucleotide has the function of a probe in ahybridization reaction to isolate or identify a polynucleotide encodinga protein comprising the amino acid sequence of SEQ ID NO:2.
 2. A vectorcomprising the isolated polynucleotide of claim
 1. 3. A method ofidentifying a nucleic acid which is at least 90% identical to thepolynucleotide of SEQ ID NO:1 and which encodes a protein having proteinkinase B activity, comprising contacting a sample with a probeconsisting of at least 26 consecutive nucleotides of SEQ ID NO:1 underconditions suitable for the nucleic acid to hybridize to the probe; anddetermining whether the nucleic acid is at least 90% identical to thepolynucleotide of SEQ ID NO:1 and encodes a polypeptide having proteinkinase B activity.
 4. An isolated polynucleotide consisting of at least32 or 35 consecutive nucleotides of SEQ ID NO:1 or the full complementof SEQ ID NO:1, wherein the isolated polynucleotide has the function ofa probe in a hybridization reaction to isolate or identify apolynucleotide encoding a protein comprising the amino acid sequence ofSEQ ID NO:2.